The present invention relates to cyclopropyl-fused pyrrolidine-based inhibitors of dipeptidyl peptidase IV (DP-4), and to a method for treating diabetes, especially Type II diabetes, as well as hyperglycemia, Syndrome X, diabetic complications, hyperinsulinemia, obesity, atherosclerosis and related diseases, as well as various immunomodulatory diseases and chronic inflammatory bowel disease, employing such cyclopropyl-fused pyrrolidines alone or in combination with another type antidiabetic agent and/or other type therapeutic agent.
Depeptidyl peptidase IV (DP-4) is a membrane bound non-classical serine aminodipeptidase which is located in a variety of tissues (intestine, liver, lung, kidney) as well as on circulating T-lymphocytes (where the enzyme is known as CD-26). It is responsible for the metabolic cleavage of certain endogenous peptides (GLP-1(7-36), glucagon) in vivo and has demonstrated proteolytic activity against a variety of other peptides (GHRH, NPY, GLP-2, VIP) in vitro.
GLP-1(7-36) is a 29 amino-acid peptide derived by post-translational processing of proglucagon in the small intestine. GLP-1(7-36) has multiple actions in vivo including the stimulation of insulin secretion, inhibition of glucagon secretion, the promotion of satiety, and the slowing of gastric emptying. Based on its physiological profile, the actions of GLP-1(7-36) are expected to be beneficial in the prevention and treatment of type II diabetes and potentially obesity. To support this claim, exogenous administration of GLP-1(7-36) (continuous infusion) in diabetic patients has demonstrated efficacy in this patient population. Unfortunately GLP-1(7-36) is degraded rapidly in vivo and has been shown to have a short half-life in vivo (t1/2≈1.5 min). Based on a study of genetically bred DP-4 KO mice and on in vivo/in vitro studies with selective DP-4 inhibitors, DP-4 has been shown to be the primary degrading enzyme of GLP-1(7-36) in vivo. GLP-1(7-36) is degraded by DP-4 efficiently to GLP-1(9-36), which has been speculated to act as a physiological antagonist to GLP-1(7-36). Thus, inhibition of DP-4 in vivo should potentiate endogenous levels of GLP-1(7-36) and attenuate formation of its antagonist GLP-1(9-36) and thus serve to ameliorate the diabetic condition.
In accordance with the present invention, cyclopropyl-fused pyrrolidine-based compounds are provided which inhibit DP-4 and have the structure 
wherein
x is 0 or 1 and y is 0 or 1 (provided that
x=1 when y=0 and
x=0 when y=1);
n is 0 or 1;
X is H or CN (that is cyano);
R1, R2, R3 and R4are the same or different and are independently selected from H, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, bicycloalkyl, tricycloalkyl, alkylcycloalkyl, hydroxyalkyl, hydroxyalkylcycloalkyl, hydroxycycloalkyl, hydroxybicycloalkyl, hydroxytricycloalkyl, bicycloalkylalkyl, alkylthioalkyl, arylalkylthioalkyl, cycloalkenyl, aryl, aralkyl, heteroaryl, heteroarylalkyl, cycloheteroalkyl and cycloheteroalkylalkyl, all optionally substituted through available carbon atoms with 1, 2, 3, 4 or 5 groups selected from hydrogen, halo, alkyl, polyhaloalkyl, alkoxy, haloalkoxy, polyhaloalkoxy, alkoxycarbonyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, polycycloalkyl, heteroarylamino, arylamino, cycloheteroalkyl, cycloheteroalkylalkyl, hydroxy, hydroxyalkyl, nitro, cyano, amino, substituted amino, alkylamino, dialkylamino, thiol, alkylthio, alkylcarbonyl, acyl, alkoxycarbonyl, aminocarbonyl, alkynylaminocarbonyl, alkylaminocarbonyl, alkenylaminocarbonyl, alkylcarbonyloxy, alkylcarbonylamino, arylcarbonylamino, alkylsulfonylamino, alkylaminocarbonylamino, alkoxycarbonylamino, alkylsulfonyl, aminosulfonyl, alkylsulfinyl, sulfonamido or sulfonyl;
and R1 and R3 may optionally be taken together to form xe2x80x94(CR5R6)mxe2x80x94 where m is 2 to 6, and R5 and R6 are the same or different and are independently selected from hydroxy, alkoxy, cyano, H, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, cycloalkenyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, cycloheteroalkyl, halo, amino, substituted amino, cycloheteroalkylalkyl, alkylcarbonylamino, arylcarbonylamino, alkoxycarbonylamino, aryloxycarbonylamino, alkoxycarbonyl, aryloxycarbonyl, or alkylaminocarbonylamino, or R1 and R4 may optionally be taken together to form xe2x80x94(CR7R8)pxe2x80x94 where p is 2 to 6, and R7 and R8 are the same or different and are independently selected from hydroxy, alkoxy, cyano, H, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, cycloalkenyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, cycloheteroalkyl, halo, amino, substituted amino, cycloheteroalkylalkyl, alkylcarbonylamino, arylcarbonylamino, alkoxycarbonylamino, aryloxycarbonylamino, alkoxycarbonyl, aryloxycarbonyl, or alkylaminocarbonylamino, or optionally R1 and R3 together with 
xe2x80x83form a 5 to 7 membered ring containing a total of 2 to 4 heteroatoms selected from N, O, S, SO, or SO2;
or optionally R1 and R3 together with 
xe2x80x83form a 4 to 8 membered cycloheteroalkyl ring wherein the cycloheteroalkyl ring has an optional aryl ring fused thereto or an optional 3 to 7 membered cycloalkyl ring fused thereto;
and including pharmaceutically acceptable salts thereof, and prodrug esters thereof, and all stereoisomers thereof.
Thus, the compounds of formula I of the invention include the following structures 
In addition, in accordance with the present invention, a method is provided for treating diabetes, especially Type II diabetes, as well as impaired glucose homeostasis, impaired glucose tolerance, infertility, polycystic ovary syndrome, growth disorders, frailty, arthritis, allograft rejection in transplantation, autoimmune diseases (such as scleroderma and multiple sclerosis), various immunomodulatory diseases (such as lupus erythematosis or psoriasis), AIDS, intestinal diseases (such as necrotizing enteritis, microvillus inclusion disease or celiac disease), inflammatory bowel syndrome, chemotherapy-induced intestinal mucosal atrophy or injury, anorexia nervosa, osteoporosis, Syndrome X, dysmetabolic syndrome, diabetic complications, hyperinsulinemia, obesity, atherosclerosis and related diseases, as well as inflammatory bowel disease (such as Crohn""s disease and ulcerative colitis), wherein a therapeutically effective amount of a compound of structure I (which inhibits DP 4) is administered to a human patient in need of treatment.
The conditions, diseases, and maladies collectively referenced to as xe2x80x9cSyndrome Xxe2x80x9d or Metabolic Syndrome are detailed in Johannsson J. Clin. Endocrinol. Metab., 82, 727-734 (1997).
In addition, in accordance with the present invention, a method is provided for treating diabetes and related diseases as defined above and hereinafter as well as any of the other disease states mentioned above, wherein a therapeutically effective amount of a combination of a compound of structure I and one, two, three or more of other types of antidiabetic agent(s) (which may be employed to treat diabetes and related diseases) and/or one, two or three or more other types of therapeutic agent(s) is administered to a human patient in need of treatment.
The term xe2x80x9cdiabetes and related diseasesxe2x80x9d refers to Type II diabetes, Type I diabetes, impaired glucose tolerance, obesity, hyperglycemia, Syndrome X, dysmetabolic syndrome, diabetic complications, dysmetabolic syndrome, and hyperinsulinemia.
The conditions, diseases and maladies collectively referred to as xe2x80x9cdiabetic complicationsxe2x80x9d include retinopathy, neuropathy and nephropathy, and other known complications of diabetes.
The term xe2x80x9cother type(s) of therapeutic agentsxe2x80x9d as employed herein refers to one or more antidiabetic agents (other than DP4 inhibitors of formula I), one or more anti-obesity agents, and/or one or more lipid-modulating agents (including anti-atherosclerosis agents), and/or one or more infertility agents, one or more agents for treating polycystic ovary syndrome, one or more agents for treating growth disorders, one or more agents for treating frailty, one or more agents for treating arthritis, one or more agents for preventing allograft rejection in transplantation, one or more agents for treating autoimmune diseases, one or more anti-AIDS agents, one or more anti-osteoporosis agents, one or more agents for treating immunomodulatory diseases, one or more agents for treating chronic inflammatory bowel disease or syndrome and/or one or more agents for treating anorexia nervosa.
The term xe2x80x9clipid-modulatingxe2x80x9d agent as employed herein refers to agents which lower LDL and/or raise HDL and/or lower triglycerides and/or lower total cholesterol and/or other known mechanisms for therapeutically treating lipid disorders.
In the above methods of the invention, the compound of structure I will be employed in a weight ratio to the antidiabetic agent or other type therapeutic agent (depending upon its mode of operation) within the range from about 0.01:1 to about 500:1, preferably from about 0.1:1 to about 100:1, more preferably from about 0.2:1 to about 10:1.
Preferred are compounds of formula I wherein R3 is H or alkyl, R1 is H, alkyl, cycloalkyl, bicycloalkyl, tricycloalkyl, alkylcycloalkyl, hydroxyalkyl, hydroxytricycloalkyl, hydroxycycloalkyl, hydroxybicycloalkyl, or hydroxyalkylcycloalkyl, R2 is H or alkyl, n is 0, X is CN, x is 0 or 1 and y is 0 or 1.
Most preferred are preferred compounds of formula I as described above where X is 
and/or wherein the fused cyclopropyl group is identified as 
Thus, preferred compounds of formula I of the invention will include the moiety: 
Particularly preferred are the following compounds: 
wherein R1 is alkyl, cycloalkyl, bicycloalkyl, tricycloalkyl, alkylcycloalkyl, hydroxyalkyl, hydroxycycloalkyl, hydroxyalkylcycloalkyl, hydroxybicycloalkyl or hydroxytricycloalkyl; 
wherein R1 is alkyl, cycloalkyl, bicycloalkyl, tricycloalkyl, hydroxybicycloalkyl, hydroxytricycloalkyl, alkylcycloalkyl, hydroxyalkyl, hydroxycycloalkyl or hydroxyalkylcycloalkyl as well as the following: 
Compounds of the structure I may be generated by the methods as shown in the following reaction schemes and the description thereof.
Referring to Reaction Scheme 1, compound 1, where PG1 is a common amine protecting group such as Boc, Cbz, or FMOC and X1 is H or CO2R9 as set out below, may be generated by methods as described herein or in the literature (for example see Sagnard et al, Tet-Lett., 1995, 36, pp. 3148-3152, Tverezovsky et al, Tetrahedron, 1997, 53, pp. 14773-14792, Hanessian et al, Bioorg. Med. Chem. Lett., 1998, 8, p. 2123-2128). Removal of the PG1 group by conventional methods (e.g. (1) TFA or HCl when PG1 is Boc, or (2) H2/Pd/C, TMSI when PG1 is Cbz, or (3) Et2NH when PG1 is (FMOC) affords the free amine 2. Amine 2 may be coupled to various protected amino acids such as 3 (where PG2 can be any of the PG1 protecting groups) using standard peptide coupling conditions (e.g. EDAC/HOAT, i-BuCOCOC1/TEA, PyBop/NMM) to afford the corresponding dipeptide 4. Removal of the amine protecting group PG2 provides compound Ia of the invention where X=H.
In the case where X1xe2x95x90CO2R9 (where R9 is alkyl or aralkyl groups such as methyl, ethyl, t-butyl, or benzyl), the ester may be hydrolyzed under a variety of conditions, for example with aqueous NaOH in a suitable solvent such as methanol, THF, or dioxane, to provide the acid 5. Conversion of the acid group to the primary carboxamide, affording 6, may be effected by activation of the acid group (e.g. employing i-BuOCOC1/TEA or EDAC) followed by treatment with NH3 or an ammonia equivalent in a solvent such as dioxane, ether, or methanol. The amide functionality may be converted to the nitrile group by a variety of standard conditions (e.g. POCl3/pyridine/imidazole or cyanuric chloride/DMF or trifluoroacetic anhydride, THF, pyridine) to give 7. Finally, removal of the PG2 protecting group similar to above provides compound of the invention Ib.
In a different sequence (Scheme 2), compound 1 where X1 is CO2R9 may be saponified to the acid and subsequently amidated as described above to give amide 8. Removal of the PG1 group followed by peptide coupling to 3 affords compound 6, an intermediate in the synthesis of Ib.
Alternately, the carboxamide group in 8 may be converted to the nitrile as described above to give compound 9. Deprotection of PGI affords 10 which may be subject to standard peptide coupling conditions to afford 7, an intermediate in the synthesis of Ib. Compound 10 may also be generated by oxidation of the amine 2 (e.g. NCS) followed by hydrolysis and subsequent cyanide treatment. Compound 10 may be obtained as a mixture of stereoisomers or a single isomer/diastereomer which may be epimerized (employing conventional procedures) to afford a mixture of stereoisomers. 
In a like manner, xcex2-amino acids such as 
may be coupled with 2, the free amine of 8, or 10 to give the corresponding amides which may be converted to the xcex2-amino acid derivatives of compound Ia or Ib following the same chemistry.
Unless otherwise indicated, the term xe2x80x9clower alkylxe2x80x9d, xe2x80x9calkylxe2x80x9d or xe2x80x9calkxe2x80x9d as employed herein alone or as part of another group includes both straight and branched chain hydrocarbons, containing 1 to 20 carbons, preferably 1 to 10 carbons, more preferably 1 to 8 carbons, in the normal chain, such as methyl, ethyl, propyl, isopropyl, butyl, t-butyl, isobutyl, pentyl, hexyl, isohexyl, heptyl, 4,4-dimethylpentyl, octyl, 2,2,4-trimethyl-pentyl, nonyl, decyl, undecyl, dodecyl, the various branched chain isomers thereof, and the like as well as such groups including 1 to 4 substituents such as halo, for example F, Br, Cl or I or CF3, alkyl, alkoxy, aryl, aryloxy, aryl(aryl) or diaryl, arylalkyl, arylalkyloxy, alkenyl, cycloalkyl, cycloalkylalkyl, cycloalkylalkyloxy, amino, hydroxy, hydroxyalkyl, acyl, heteroaryl, heteroaryloxy, heteroarylalkyl, heteroarylalkoxy, aryloxyalkyl, alkylthio, arylalkylthio, aryloxyaryl, alkylamido, alkanoylamino, arylcarbonylamino, nitro, cyano, thiol, haloalkyl, trihaloalkyl and/or alkylthio.
Unless otherwise indicated, the term xe2x80x9ccycloalkylxe2x80x9d as employed herein alone or as part of another group includes saturated or partially unsaturated (containing 1 or 2 double bonds) cyclic hydrocarbon groups containing 1 to 3 rings, including monocyclic alkyl, bicyclic alkyl (or bicycloalkyl) and tricyclic alkyl (tricycloalkyl), containing a total of 3 to 20 carbons forming the ring, preferably 3 to 10 carbons, forming the ring and which may be fused to 1 or 2 aromatic rings as described for aryl, which includes cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclodecyl and cyclododecyl, cyclohexenyl, adamantyl, 
any of which groups may be optionally substituted with 1 to 4 substituents such as halogen, alkyl, alkoxy, hydroxy, aryl, aryloxy, arylalkyl, cycloalkyl, hydroxyalkyl, alkylamido, alkanoylamino, oxo, acyl, arylcarbonylamino, amino, nitro, cyano, thiol and/or alkylthio and/or any of the substituents for alkyl.
The term xe2x80x9ccycloalkenylxe2x80x9d as employed herein alone or as part of another group refers to cyclic hydrocarbons containing 3 to 12 carbons, preferably 5 to 10 carbons and 1 or 2 double bonds. Exemplary cycloalkenyl groups include cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl, cyclohexadienyl, and cycloheptadienyl, which may be optionally substituted as defined for cycloalkyl.
The term xe2x80x9ccycloalkylenexe2x80x9d as employed herein refers to a xe2x80x9ccycloalkylxe2x80x9d group which includes free bonds and thus is a linking group such as 
and the like, and may optionally be substituted as defined above for xe2x80x9ccycloalkylxe2x80x9d.
The term xe2x80x9calkanoylxe2x80x9d as used herein alone or as part of another group refers to alkyl linked to a carbonyl group.
Unless otherwise indicated, the term xe2x80x9clower alkenylxe2x80x9d or xe2x80x9calkenylxe2x80x9d as used herein by itself or as part of another group refers to straight or branched chain radicals of 2 to 20 carbons, preferably 2 to 12 carbons, and more preferably 1 to 8 carbons in the normal chain, which include one to six double bonds in the normal chain, such as vinyl, 2-propenyl, 3-butenyl, 2-butenyl, 4-pentenyl, 3-pentenyl, 2-hexenyl, 3-hexenyl, 2-heptenyl, 3-heptenyl, 4-heptenyl, 3-octenyl, 3-nonenyl, 4-decenyl, 3-undecenyl, 4-dodecenyl, 4,8,12-tetradecatrienyl, and the like, and which may be optionally substituted with 1 to 4 substituents, namely, halogen, haloalkyl, alkyl, alkoxy, alkenyl, alkynyl, aryl, arylalkyl, cycloalkyl, amino, hydroxy, heteroaryl, cycloheteroalkyl, alkanoylamino, alkylamido, arylcarbonyl-amino, nitro, cyano, thiol, alkylthio and/or any of the alkyl substituents set out herein.
Unless otherwise indicated, the term xe2x80x9clower alkynylxe2x80x9d or xe2x80x9calkynylxe2x80x9d as used herein by itself or as part of another group refers to straight or branched chain radicals of 2 to 20 carbons, preferably 2 to 12 carbons and more preferably 2 to 8 carbons in the normal chain, which include one triple bond in the normal chain, such as 2-propynyl, 3-butynyl, 2-butynyl, 4-pentynyl, 3-pentynyl, 2-hexynyl, 3-hexynyl, 2-heptynyl, 3-heptynyl, 4-heptynyl, 3-octenyl, 3-nonenyl, 4-decenyl,3-undecenyl, 4-dodecenyl and the like, and which may be optionally substituted with 1 to 4 substituents, namely, halogen, haloalkyl, alkyl, alkoxy, alkenyl, alkynyl, aryl, arylalkyl, cycloalkyl, amino, heteroaryl, cycloheteroalkyl, hydroxy, alkanoylamino, alkylamido, arylcarbonylamino, nitro, cyano, thiol, and/or alkylthio, and/or any of the alkyl substituents set out herein.
The terms xe2x80x9carylalkenylxe2x80x9d and xe2x80x9carylalkynylxe2x80x9d as used alone or as part of another group refer to alkenyl and alkynyl groups as described above having an aryl substituent.
Where alkyl groups as defined above have single bonds for attachment to other groups at two different carbon atoms, they are termed xe2x80x9calkylenexe2x80x9d groups and may optionally be substituted as defined above for xe2x80x9calkylxe2x80x9d.
Where alkenyl groups as defined above and alkynyl groups as defined above, respectively, have single bonds for attachment at two different carbon atoms, they are termed xe2x80x9calkenylene groupsxe2x80x9d and xe2x80x9calkynylene groupsxe2x80x9d, respectively, and may optionally be substituted as defined above for xe2x80x9calkenylxe2x80x9d and xe2x80x9calkynylxe2x80x9d.
The term xe2x80x9chalogenxe2x80x9d or xe2x80x9chaloxe2x80x9d as used herein alone or as part of another group refers to chlorine, bromine, fluorine, and iodine as well as CF3, with chlorine or fluorine being preferred.
The term xe2x80x9cmetal ionxe2x80x9d refers to alkali metal ions such as sodium, potassium or lithium and alkaline earth metal ions such as magnesium and calcium, as well as zinc and aluminum.
Unless otherwise indicated, the term xe2x80x9carylxe2x80x9d as employed herein alone or as part of another group refers to monocyclic and bicyclic aromatic groups containing 6 to 10 carbons in the ring portion (such as phenyl or naphthyl including 1-naphthyl and 2-naphthyl) and may optionally include one to three additional rings fused to a carbocyclic ring or a heterocyclic ring (such as aryl, cycloalkyl, heteroaryl or cycloheteroalkyl rings for example 
and may be optionally substituted through available carbon atoms with 1, 2, or 3 groups selected from hydrogen, halo, haloalkyl, alkyl, haloalkyl, alkoxy, haloalkoxy, alkenyl, trifluoromethyl, trifluoromethoxy, alkynyl, cycloalkylalkyl, cycloheteroalkyl, cycloheteroalkylalkyl, aryl, heteroaryl, arylalkyl, aryloxy, aryloxyalkyl, arylalkoxy, arylthio, arylazo, heteroarylalkyl, heteroarylalkenyl, heteroarylheteroaryl, heteroaryloxy, hydroxy, nitro, cyano, amino, substituted amino wherein the amino includes 1 or 2 substituents (which are alkyl, aryl or any of the other aryl compounds mentioned in the definitions), thiol, alkylthio, arylthio, heteroarylthio, arylthioalkyl, alkoxyarylthio, alkylcarbonyl, arylcarbonyl, alkylaminocarbonyl, arylaminocarbonyl, alkoxycarbonyl, aminocarbonyl, alkylcarbonyloxy, arylcarbonyloxy, alkylcarbonylamino, arylcarbonylamino, arylsulfinyl, arylsulfinylalkyl, arylsulfonylamino or arylsulfon-aminocarbonyl and/or any of the alkyl substituents set out herein.
Unless otherwise indicated, the term xe2x80x9clower alkoxyxe2x80x9d, xe2x80x9calkoxyxe2x80x9d, xe2x80x9caryloxyxe2x80x9d or xe2x80x9caralkoxyxe2x80x9d as employed herein alone or as part of another group includes any of the above alkyl, aralkyl or aryl groups linked to an oxygen atom.
Unless otherwise indicated, the term xe2x80x9csubstituted aminoxe2x80x9d as employed herein alone or as part of another group refers to amino substituted with one or two substituents, which may be the same or different, such as alkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, cycloheteroalkyl, cycloheteroalkylalkyl, cycloalkyl, cycloalkylalkyl haloalkyl, hydroxyalkyl, alkoxyalkyl or thioalkyl. These substituents may be further substituted with any of the R1 groups or substituents for R1 as set out above. In addition, the amino substituents may be taken together with the nitrogen atom to which they are attached to form 1-pyrrolidinyl, 1-piperidinyl, 1-azepinyl, 4-morpholinyl, 4-thiamorpholinyl, 1-piperazinyl, 4-alkyl-l-piperazinyl, 4-arylalkyl-1-piperazinyl, 4-diarylalkyl-l-piperazinyl, 1-pyrrolidinyl, 1-piperidinyl, or 1-azepinyl, optionally substituted with alkyl, alkoxy, alkylthio, halo, trifluoromethyl or hydroxy.
Unless otherwise indicated, the term xe2x80x9clower alkylthioxe2x80x9d, xe2x80x9calkylthioxe2x80x9d, xe2x80x9carylthioxe2x80x9d or xe2x80x9caralkylthioxe2x80x9d as employed herein alone or as part of another group includes any of the above alkyl, aralkyl or aryl groups linked to a sulfur atom.
Unless otherwise indicated, the term xe2x80x9clower alkylaminoxe2x80x9d, xe2x80x9calkylaminoxe2x80x9d, xe2x80x9carylaminoxe2x80x9d, or xe2x80x9carylalkylaminoxe2x80x9d as employed herein alone or as part of another group includes any of the above alkyl, aryl or arylalkyl groups linked to a nitrogen atom.
Unless otherwise indicated, the term xe2x80x9cacylxe2x80x9d as employed herein by itself or part of another group, as defined herein, refers to an organic radical linked to a carbonyl 
group; examples of acyl groups include any of the R1 groups attached to a carbonyl, such as alkanoyl, alkenoyl, aroyl, aralkanoyl, heteroaroyl, cycloalkanoyl, cycloheteroalkanoyl and the like.
Unless otherwise indicated, the term xe2x80x9ccycloheteroalkylxe2x80x9d as used herein alone or as part of another group refers to a 5-, 6- or 7-membered saturated or partially unsaturated ring which includes 1 to 2 hetero atoms such as nitrogen, oxygen and/or sulfur, linked through a carbon atom or a heteroatom, where possible, optionally via the linker (CH2)r (where r is 1, 2 or 3), such as: 
and the like. The above groups may include 1 to 4 substituents such as alkyl, halo, oxo and/or any of the alkyl substituents set out herein. In addition, any of the cycloheteroalkyl rings can be fused to a cycloalkyl, aryl, heteroaryl or cycloheteroalkyl ring.
Unless otherwise indicated, the term xe2x80x9cheteroarylxe2x80x9d as used herein alone or as part of another group refers to a 5- or 6- membered aromatic ring which includes 1, 2, 3 or 4 hetero atoms such as nitrogen, oxygen or sulfur, and such rings fused to an aryl, cycloalkyl, heteroaryl or cycloheteroalkyl ring (e.g. benzothiophenyl, indolyl), and includes possible N-oxides. The heteroaryl group may optionally include 1 to 4 substituents such as any of the substituents set out above for alkyl. Examples of heteroaryl groups include the following: 
and the like.
The term xe2x80x9ccycloheteroalkylalkylxe2x80x9d as used herein alone or as part of another group refers cycloheteroalkyl groups as defined above linked through a atom or heteroatom to a (CH2)r chain.
The term xe2x80x9cheteroarylalkylxe2x80x9d or xe2x80x9cheteroarylalkenylxe2x80x9d as used herein alone or as part of another group refers to a heteroaryl group as defined above linked through a C atom or heteroatom to a xe2x80x94(CH2)rxe2x80x94 chain, alkylene or alkenylene as defined above.
The term xe2x80x9cpolyhaloalkylxe2x80x9d as used herein refers to an xe2x80x9calkylxe2x80x9d group as defined above which includes from 2 to 9, preferably from 2 to 5, halo substituents, such as F or Cl, preferably F, such as CF3CH2, CF3 or CF3CF2CH2.
The term xe2x80x9cpolyhaloalkoxyxe2x80x9d as used herein refers to an xe2x80x9calkoxyxe2x80x9d or xe2x80x9calkyloxyxe2x80x9d group as defined above which includes from 2 to 9, preferably from 2 to 5, halo substituents, such as F or Cl, preferably F, such as CF3CH2O, CF3O or CF3CF2CH2O.
All stereoisomers of the compounds of the instant invention are contemplated, either in admixture or in pure or substantially pure form. The compounds of the present invention can have asymmetric centers at any of the carbon atoms including any one or the R substituents. Consequently, compounds of formula I can exist in enantiomeric or diastereomeric forms or in mixtures thereof. The processes for preparation can utilize racemates, enantiomers or diastereomers as starting materials. When diastereomeric or enantiomeric products are prepared, they can be separated by conventional methods for example, chromatographic or fractional crystallization.
Where desired, the compounds of structure I may be used in combination with one or more other types of antidiabetic agents (employed to treat diabetes and related diseases) and/or one or more other types of therapeutic agents which may be administered orally in the same dosage form, in a separate oral dosage form or by injection.
The other type of antidiabetic agent which may be optionally employed in combination with the DP4 inhibitor of formula I may be 1,2,3 or more antidiabetic agents or antihyperglycemic agents including insulin secretagogues or insulin sensitizers, or other antidiabetic agents preferably having a mechanism of action different from DP4 inhibition and may include biguanides, sulfonyl ureas, glucosidase inhibitors, PPAR xcex3 agonists, such as thiazolidinediones, SGLT2 inhibitors, PPAR xcex1/xcex3 dual agonists, aP2 inhibitors, glycogen phosphorylase inhibitors, advanced glycosylation end (AGE) products inhibitors, and/or meglitinides, as well as insulin, and/or glucagon-like peptide-1 (GLP-1) or mimetics thereof.
It is believed that the use of the compounds of structure I in combination with 1, 2, 3 or more other antidiabetic agents produces antihyperglycemic results greater than that possible from each of these medicaments alone and greater than the combined additive antihyperglycemic effects produced by these medicaments.
The other antidiabetic agent may be an oral antihyperglycemic agent preferably a biguanide such as metformin or phenformin or salts thereof, preferably metformin HCl.
Where the other antidiabetic agent is a biguanide, the compounds of structure I will be employed in a weight ratio to biguanide within the range from about 0.01:1 to about 100:1, preferably from about 0.1:1 to about 5:1.
The other antidiabetic agent may also preferably be a sulfonyl urea such as glyburide (also known as glibenclamide), glimepiride (disclosed in U.S. Pat. No. 4,379,785), glipizide, gliclazide or chlorpropamide, other known sulfonylureas or other antihyperglycemic agents which act on the ATP-dependent channel of the xcex3-cells, with glyburide and glipizide being preferred, which may be administered in the same or in separate oral dosage forms.
The compounds of structure I will be employed in a weight ratio to the sulfonyl urea in the range from about 0.01:1 to about 100:1, preferably from about 0.05:1 to about 5:1.
The oral antidiabetic agent may also be a glucosidase inhibitor such as acarbose (disclosed in U.S. Pat. No. 4,904,769) or miglitol (disclosed in U.S. Pat. No. 4,639,436), which may be administered in the same or in a separate oral dosage forms.
The compounds of structure I will be employed in a weight ratio to the glucosidase inhibitor within the range from about 0.01:1 to about 100:1, preferably from about 0.2:1 to about 50:1.
The compounds of structure I may be employed in combination with a PPAR xcex3 agonist such as a thiazolidinedione oral anti-diabetic agent or other insulin sensitizers (which has an insulin sensitivity effect in NIDDM patients) such as troglitazone (Warner-Lambert""s Rezulin(copyright), disclosed in U.S. Pat. No. 4,572,912), rosiglitazone (en), pioglitazone (Takeda), Mitsubishi MCC-555 (disclosed in U.S. Pat. No. 5,594,016), Glaxo-Wellcome""s GL-262570, englitazone (CP-68722, Pfizer) or darglitazone (CP-86325, Pfizer, isaglitazone (MIT/JandJ), JTT-501 (JPNT/PandU), L-895645 (Merck), R-119702 (Sankyo/WL), NN-2344 (Dr. Reddy/NN), or YM-440 (Yamanouchi), preferably rosiglitazone and pioglitazone.
The compounds of structure I will be employed in a weight ratio to the thiazolidinedione in an amount within the range from about 0.01:1 to about 100:1, preferably from about 0.1:1 to about 10:1.
The sulfonyl urea and thiazolidinedione in amounts of less than about 150 mg oral antidiabetic agent may be incorporated in a single tablet with the compounds of structure I.
The compounds of structure I may also be employed in combination with a antihyperglycemic agent such as insulin or with glucagon-like peptide-1 (GLP-1) such as GLP-1(1-36) amide, GLP-1(7-36) amide, GLP-1(7-36) (as disclosed in U.S. Pat. No. 5,614,492 to Habener, disclosure of which is incorporated herein by reference), or a GLP-1 mimic such as AC2993 or Exendin-4 (Amylin) and LY-315902 or LY-307167 (Lilly) and NN2211 (Novo-Nordisk), which may be administered via injection, intranasal, or by transdermal or buccal devices.
Where present, metformin, the sulfonyl ureas, such as glyburide, glimepiride, glipyride, glipizide, chlorpropamide and gliclazide and the glucosidase inhibitors acarbose or miglitol or insulin (injectable, pulmonary, buccal, or oral) may be employed in formulations as described above and in amounts and dosing as indicated in the Physician""s Desk Reference (PDR).
Where present, metformin or salt thereof may be employed in amounts within the range from about 500 to about 2000 mg per day which may be administered in single or divided doses one to four times daily.
Where present, the thiazolidinedione anti-diabetic agent may be employed in amounts within the range from about 0.01 to about 2000 mg/day which may be administered in single or divided doses one to four times per day.
Where present insulin may be employed in formulations, amounts and dosing as indicated by the Physician""s Desk Reference.
Where present GLP-1 peptides may be administered in oral buccal formulations, by nasal administration (for example inhalation spray) or parenterally as described in U.S. Pat. Nos. 5,346,701 (TheraTech), 5,614,492 and 5,631,224 which are incorporated herein by reference.
The other antidiabetic agent may also be a PPAR xcex1/xcex3 dual agonist such as AR-HO39242 (Astra/Zeneca), GW-409544 (Glaxo-Wellcome), KRP297 (Kyorin Merck) as well as those disclosed by Murakami et al, xe2x80x9cA Novel Insulin Sensitizer Acts As a Coligand for Peroxisome Proliferationxe2x80x94Activated Receptor Alpha (PPAR alpha) and PPAR gamma. Effect on PPAR alpha Activation on Abnormal Lipid Metabolism in Liver of Zucker Fatty Ratsxe2x80x9d, Diabetes 47, 1841-1847 (1998), and in U.S. application Ser. No. 09/664,598, filed Sep. 18, 2000, (attorney file LA29NP) the disclosure of which is incorporated herein by reference, employing dosages as set out therein, which compounds designated as preferred are preferred for use herein.
The other antidiabetic agent may be an SGLT2 inhibitor such as disclosed in U.S. application Ser. No. 09/679,027, filed Oct. 4, 2000 (attorney file LA49NP), which is incorporated herein by reference, employing dosages as set out herein. Preferred are the compounds designated as preferred in the above application.
The other antidiabetic agent which may be optionally employed in combination with the DP4 inhibitor of formula I may be an aP2 inhibitor such as disclosed in U.S. application Ser. No. 09/391,053, filed Sep. 7, 1999, and U.S. application Ser. No. 09/519,079, filed Mar. 6, 2000 (attorney file LA27NP), which is incorporated herein by reference, employing dosages as set out herein. Preferred are the compounds designated as preferred in the above application.
The other antidiabetic agent which may be optionally employed in combination with the DP4 inhibitor of formula I may be a glycogen phosphorylase inhibitor such as disclosed in WO 96/39384, WO 96/39385, EP 978279, WO 2000/47206, WO 99/43663, and U.S. Pat. Nos. 5,952,322 and 5,998,463, WO 99/26659 and EP 1041068.
The meglitinide which may optionally be employed in combination with the compound of formula I of the invention may be repaglinide, nateglinide (Novartis) or KAD1229 (PF/Kissei), with repaglinide being preferred.
The DP4 inhibitor of formula I will be employed in a weight ratio to the meglitinide, PPAR xcex3 agonist, PPAR xcex1/xcex3 dual agonist, SGLT2 inhibitor, aP2 inhibitor, or glycogen phosphorylase inhibitor within the range from about 0.01:1 to about 100:1, preferably from about 0.1:1 to about 10:1.
The hypolipidemic agent or lipid-modulating agent which may be optionally employed in combination with the compounds of formula I of the invention may include 1,2,3 or more MTP inhibitors, HMG CoA reductase inhibitors, squalene synthetase inhibitors, fibric acid derivatives, ACAT inhibitors, lipoxygenase inhibitors, cholesterol absorption inhibitors, ileal Na+/bile acid cotransporter inhibitors, upregulators of LDL receptor activity, ATP citrate lyase inhibitors, cholesteryl ester transfer protein inhibitors, bile acid sequestrants, and/or nicotinic acid and derivatives thereof.
MTP inhibitors employed herein include MTP inhibitors disclosed in U.S. Pat. No. 5,595,872, U.S. Pat. No. 5,739,135, U.S. Pat. No. 5,712,279, U.S. Pat. No. 5,760,246, U.S. Pat. No. 5,827,875, U.S. Pat. No. 5,885,983 and U.S. application Ser. No. 09/175,180 filed Oct. 20, 1998, now U.S. Pat. No. 5,962,440. Preferred are each of the preferred MTP inhibitors disclosed in each of the above patents and applications.
All of the above U.S. Patents and applications are incorporated herein by reference.
Most preferred MTP inhibitors to be employed in accordance with the present invention include preferred MTP inhibitors as set out in U.S. Pat. Nos. 5,739,135 and 5,712,279, and U.S. Pat. No. 5,760,246 as well as implitapide (Bayer).
The most preferred MTP inhibitor is 9-[4-[4-[[2-(2,2,2-Trifluoroethoxy)benzoyl]amino]-1-piperidinyl] butyl]-N-(2,2,2-trifluoroethyl)-9H-fluorene-9-carboxamide 
The hypolipidemic agent may be an HMG CoA reductase inhibitor which includes, but is not limited to, mevastatin and related compounds as disclosed in U.S. Pat. No. 3,983,140, lovastatin (mevinolin) and related compounds as disclosed in U.S. Pat. No. 4,231,938, pravastatin and related compounds such as disclosed in U.S. Pat. No. 4,346,227, simvastatin and related compounds as disclosed in U.S. Pat. Nos. 4,448,784 and 4,450,171. Other HMG CoA reductase inhibitors which may be employed herein include, but are not limited to, fluvastatin, disclosed in U.S. Pat. No. 5,354,772, cerivastatin disclosed in U.S. Pat. Nos. 5,006,530 and 5,177,080, atorvastatin disclosed in U.S. Pat. Nos. 4,681,893, 5,273,995, 5,385,929 and 5,686,104, atavastatin (Nissan/Sankyo nisvastatin (NK-104)) disclosed in U.S. Pat. No. 5,011,930, Shionogi-Astra/Zeneca visastatin (ZD-4522) disclosed in U.S. Patent No. 5,260,440.
The squalene synthetase inhibitors suitable for use herein include, but are not limited to, xcex1-phosphono-sulfonates disclosed in U.S. Pat. No. 5,712,396, those disclosed by Biller et al, J. Med. Chem., 1988, Vol. 11, No. 10, pp 1869-1871, including isoprenoid (phosphinyl-methyl)phosphonates as well as other known squalene synthetase inhibitors, for example, as disclosed in U.S. Pat. Nos. 4,871,721 and 4,924,024 and in Biller, S. A., Neuenschwander, K., Ponpipom, M. M., and Poulter, C. D., Current Pharmaceutical Design, 2, 1-40 (1996).
In addition, other squalene synthetase inhibitors suitable for use herein include the terpenoid pyrophosphates disclosed by P. Ortiz de Montellano et al, J. Med. Chem., 1977, 20, 243-249, the farnesyl diphosphate analog A and presqualene pyrophosphate (PSQ-PP) analogs as disclosed by Corey and Volante, J. Am. Chem. Soc., 1976, 98, 1291-1293, phosphinylphosphonates reported by McClard, R. W. et al, J.A.C.S., 1987, 10, 5544 and cyclopropanes reported by Capson, T. L., PhD dissertation, June, 1987, Dept. Med. Chem. U of Utah, Abstracts Table of Contents, pp 16, 17, 40-43, 48-51, Summary.
Other hypolipidemic agents suitable for use herein include, but are not limited to, fibric acid derivatives, such as fenofibrate, gemfibrozil, clofibrate, bezafibrate, ciprofibrate, clinofibrate and the like, probucol, and related compounds as disclosed in U.S. Pat. No. 3,674,836, probucol and gemfibrozil being preferred, bile acid sequestrants such as cholestyramine, colestipol and DEAE-Sephadex (Secholex(copyright), Policexide(copyright)), as well as lipostabil (Rhone-Poulenc), Eisai E-5050 (an N-substituted ethanolamine derivative), imanixil (HOE-402), tetrahydrolipstatin (THL), istigmastanylphos-phorylcholine (SPC, Roche), aminocyclodextrin (Tanabe Seiyoku), Ajinomoto AJ-814 (azulene derivative), melinamide (Sumitomo), Sandoz 58-035, American Cyanamid CL-277,082 and CL-283,546 (disubstituted urea derivatives), nicotinic acid, acipimox, acifran, neomycin, p-aminosalicylic acid, aspirin, poly(diallylmethylamine) derivatives such as disclosed in U.S. Pat. No. 4,759,923, quaternary amine poly(diallyldimethylammonium chloride) and ionenes such as disclosed in U.S. Pat. No. 4,027,009, and other known serum cholesterol lowering agents.
The other hypolipidemic agent may be an ACAT inhibitor such as disclosed in, Drugs of the Future 24, 9-15 (1999), (Avasimibe); xe2x80x9cThe ACAT inhibitor, Cl-1011 is effective in the prevention and regression of aortic fatty streak area in hamstersxe2x80x9d, Nicolosi et al, Atherosclerosis (Shannon, Irel). (1998), 137(1), 77-85; xe2x80x9cThe pharmacological profile of FCE 27677: a novel ACAT inhibitor with potent hypolipidemic activity mediated by selective suppression of the hepatic secretion of ApoB100-containing lipoproteinxe2x80x9d, Ghiselli, Giancarlo, Cardiovasc. Drug Rev. (1998), 16(1), 16-30; xe2x80x9cRP 73163: a bioavailable alkylsulfinyl-diphenylimidazole ACAT inhibitorxe2x80x9d, Smith, C., et al, Bioorg. Med. Chem. Lett. (1996), 6(1), 47-50; xe2x80x9cACAT inhibitors: physiologic mechanisms for hypolipidemic and anti-atherosclerotic activities in experimental animalsxe2x80x9d, Krause et al, Editor(s): Ruffolo, Robert R., Jr.; Hollinger, Mannfred A., Inflammation: Mediators Pathways (1995), 173-98, Publisher: CRC, Boca Raton, Fla.; xe2x80x9cACAT inhibitors: potential anti-atherosclerotic agentsxe2x80x9d, Sliskovic et al, Curr. Med. Chem. (1994), 1(3), 204-25; xe2x80x9cInhibitors of acyl-CoA:cholesterol O-acyl transferase (ACAT) as hypocholesterolemic agents. 6. The first water-soluble ACAT inhibitor with lipid-regulating activity. Inhibitors of acyl-CoA:cholesterol acyltransferase (ACAT). 7. Development of a series of substituted N-phenyl-Nxe2x80x2-[(1-phenylcyclopentyl)methyl]ureas with enhanced hypocholesterolemic activityxe2x80x9d, Stout et al, Chemtracts: Org. Chem. (1995), 8(6), 359-62, or TS-962 (Taisho Pharmaceutical Co. Ltd).
The hypolipidemic agent may be an upregulator of LD2 receptor activity such as MD-700 (Taisho Pharmaceutical Co. Ltd) and LY295427 (Eli Lilly).
The hypolipidemic agent may be a cholesterol absorption inhibitor preferably Schering-Plough""s SCH48461 as well as those disclosed in Atherosclerosis 115, 45-63 (1995) and J. Med. Chem. 41, 973 (1998).
The hypolipidemic agent may be an ileal Na+/bile acid cotransporter inhibitor such as disclosed in Drugs of the Future, 24, 425-430 (1999).
The lipid-modulating agent may be a cholesteryl ester transfer protein (CETP) inhibitor such as Pfizer""s CP 529,414 (WO/0038722 and EP 818448) and Pharmacia""s SC-744 and SC-795.
The ATP citrate lyase inhibitor which may be employed in the combination of the invention may include, for example, those disclosed in U.S. Pat. No. 5,447,954.
Preferred hypolipidemic agents are pravastatin, lovastatin, simvastatin, atorvastatin, fluvastatin, cerivastatin, atavastatin and ZD-4522.
The above-mentioned U.S. patents are incorporated herein by reference. The amounts and dosages employed will be as indicated in the Physician""s Desk Reference and/or in the patents set out above.
The compounds of formula I of the invention will be employed in a weight ratio to the hypolipidemic agent (were present), within the range from about 500:1 to about 1:500, preferably from about 100:1 to about 1:100.
The dose administered must be carefully adjusted according to age, weight and condition of the patient, as well as the route of administration, dosage form and regimen and the desired result.
The dosages and formulations for the hypolipidemic agent will be as disclosed in the various patents and applications discussed above.
The dosages and formulations for the other hypolipidemic agent to be employed, where applicable, will be as set out in the latest edition of the Physicians"" Desk Reference.
For oral administration, a satisfactory result may be obtained employing the MTP inhibitor in an amount within the range of from about 0.01 mg/kg to about 500 mg and preferably from about 0.1 mg to about 100 mg, one to four times daily.
A preferred oral dosage form, such as tablets or capsules, will contain the MTP inhibitor in an amount of from about 1 to about 500 mg, preferably from about 2 to about 400 mg, and more preferably from about 5 to about 250 mg, one to four times daily.
For oral administration, a satisfactory result may be obtained employing an HMG CoA reductase inhibitor, for example, pravastatin, lovastatin, simvastatin, atorvastatin, fluvastatin or cerivastatin in dosages employed as indicated in the Physician""s Desk Reference, such as in an amount within the range of from about 1 to 2000 mg, and preferably from about 4 to about 200 mg.
The squalene synthetase inhibitor may be employed in dosages in an amount within the range of from about 10 mg to about 2000 mg and preferably from about 25 mg to about 200 mg.
A preferred oral dosage form, such as tablets or capsules, will contain the HMG CoA reductase inhibitor in an amount from about 0.1 to about 100 mg, preferably from about 5 to about 80 mg, and more preferably from about 10 to about 40 mg.
A preferred oral dosage form, such as tablets or capsules will contain the squalene synthetase inhibitor in an amount of from about 10 to about 500 mg, preferably from about 25 to about 200 mg.
The other hypolipidemic agent may also be a lipoxygenase inhibitor including a 15-lipoxygenase (15-LO) inhibitor such as benzimidazole derivatives as disclosed in WO 97/12615, 15-LO inhibitors as disclosed in WO 97/12613, isothiazolones as disclosed in WO 96/38144, and 15-LO inhibitors as disclosed by Sendobry et al xe2x80x9cAttenuation of diet-induced atherosclerosis in rabbits with a highly selective 15-lipoxygenase inhibitor lacking significant antioxidant propertiesxe2x80x9d, Brit. J. Pharmacology (1997) 120, 1199-1206, and Cornicelli et al, xe2x80x9c15-Lipoxygenase and its Inhibition: A Novel Therapeutic Target for Vascular Diseasexe2x80x9d, Current Pharmaceutical Design, 1999, 5, 11-20.
The compounds of formula I and the hypolipidemic agent may be employed together in the same oral dosage form or in separate oral dosage forms taken at the same time.
The compositions described above may be administered in the dosage forms as described above in single or divided doses of one to four times daily. It may be advisable to start a patient on a low dose combination and work up gradually to a high dose combination.
The preferred hypolipidemic agent is pravastatin, simvastatin, lovastatin, atorvastatin, fluvastatin or cerivastatin.
The other type of therapeutic agent which may be optionally employed with the DP4 inhibitor of formula I may be 1, 2, 1 or more of an anti-obesity agent including a beta 3 adrenergic agonist, a lipase inhibitor, a serotonin (and dopamine) reuptake inhibitor, a thyroid receptor beta drug, an anorectic agent and/or a fatty acid oxidation upregulator.
The beta 3 adrenergic agonist which may be optionally employed in combination with a compound of formula I may be AJ9677 (Takeda/Dainippon), L750355 (Merck), or CP331648 (Pfizer) or other known beta 3 agonists as disclosed in U.S. Pat. Nos. 5,541,204, 5,770,615, 5,491,134, 5,776,983 and 5,488,064, with AJ9677, L750,355 and CP331648 being preferred.
The lipase inhibitor which may be optionally employed in combination with a compound of formula I may be orlistat or ATL-962 (Alizyme), with orlistat being preferred.
The serotonin (and dopoamine) reuptake inhibitor which may be optionally employed in combination with a compound of formula I may be sibutramine, topiramate (Johnson and Johnson) or axokine (Regeneron), with sibutramine and topiramate being preferred.
The thyroid receptor beta compound which may be optionally employed in combination with a compound of formula I may be a thyroid receptor ligand as disclosed in WO97/21993 (U. Cal SF), WO099/00353 (KaroBio) and GB98/284425 (KaroBio), with compounds of the KaroBio applications being preferred.
The anorectic agent which may be optionally employed in combination with a compound of formula I may be dexamphetamine, phentermine, phenylpropanolamine or mazindol, with dexamphetamine being preferred.
The fatty acid oxidation upregulator which may be optionally employed in combination with the compound of formula I can be famoxin (Genset).
The various anti-obesity agents described above may be employed in the same dosage form with the compound of formula I or in different dosage forms, in dosages and regimens as generally known in the art or in the PDR.
The infertility agent which may be optionally employed in combination with the DP4 inhibitor of the invention may be 1, 2, or more of clomiphene citrate (Clomid(copyright), Aventis), bromocriptine mesylate (Parlodel(copyright), Novartis),LHRH analogs, Lupron (TAP Pharm.), danazol, Danocrine (Sanofi), progestogens or glucocorticoids, which may be employed in amounts specified in the PDR.
The agent for polycystic ovary syndrome which may be optionally employed in combination with the DP4 inhibitor of the invention may be 1, 2, or more of gonadotropin releasing hormone (GnRH), leuprolide (Lupron(copyright)), Clomid(copyright), Parlodel(copyright), oral contraceptives or insulin sensitizers such as PPAR agonists, or other conventional agents for such use which may be employed in amounts specified in the PDR.
The agent for treating growth disorders and/or frailty which may be optionally employed in combination with the DP4 inhibitor of the invention may be 1, 2, or more of a growth hormone or growth hormone secretagogue such as MK-677 (Merck), CP-424,391 (Pfizer), and compounds disclosed in U.S. Ser. No. 09/506,749 filed Feb. 18, 2000 (attorney docket LA26), as well as selective androgen receptor modulators (SARMs), which is incorporated herein by reference, which may be employed in amounts specified in the PDR, where applicable.
The agent for treating arthritis which may be optionally employed in combination with the DP4 inhibitor of the invention may be 1, 2, or more of aspirin, indomethacin, ibuprofen, diclofenac sodium, naproxen, nabumetone (Relafen(copyright), SmithKline Beecham), tolmetin sodium (Tolectin(copyright), Ortho-McNeil), piroxicam (Feldene(copyright), Pfizer), ketorolac tromethamine (Toradol(copyright), Roche), celecoxib (Celebrex(copyright), Searle), rofecoxib (Vioxx(copyright), Merck) and the like, which may be employed in amounts specified in the PDR.
Conventional agents for preventing allograft rejection in transplantation such as cyclosporin, Sandimmune (Novartis), azathioprine, Immuran (Faro) or methotrexate may be optionally employed in combination with the DP4 inhibitor of the invention, which may be employed in amounts specified in the PDR.
Conventional agents for treating autoimmune diseases such as multiple sclerosis and immunomodulatory diseases such as lupus erythematosis, psoriasis, for example, azathioprine, Immuran, cyclophosphamide, NSAIDS such as ibuprofen, cox 2 inhibitors such as Vioxx and Celebrex, glucocorticoids and hydroxychloroquine, may be optionally employed in combination with the DP4 inhibitor of the invention, which may be employed in amounts specified in the PDR.
The AIDS agent which may be optionally employed in combination with the DP4 inhibitor of the invention may be a non-nucleoside reverse transcriptase inhibitor, a nucleoside reverse transcriptase inhibitor, a protease inhibitor and/or an AIDS adjunct anti-infective and may be 1, 2, or more of dronabinol (Marinol(copyright), Roxane Labs), didanosine (Videx(copyright), Bristol-Myers Squibb), megestrol acetate (Megace(copyright), Bristol-Myers Squibb), stavudine (Zerit(copyright), Bristol-Myers Squibb), delavirdine mesylate (Rescriptor(copyright), Pharmacia), lamivudine/zidovudine (Combivir(trademark), Glaxo), lamivudine (Epivir(trademark), Glaxo), zalcitabine (Hivid(copyright), Roche), zidovudine (Retrovir(copyright), Glaxo), indinavir sulfate (Crixivan(copyright), Merck), saquinavir (Fortovase(trademark), Roche), saquinovir mesylate (Invirase(copyright), Roche), ritonavir (Norvir(copyright), Abbott), nelfinavir (Viracept(copyright), Agouron).
The above anti-AIDS agents may be employed in amounts specified in the PDR.
The agent for treating inflammatory bowel disease or syndrome which may be optionally employed in combination with the DP4 inhibitor of the invention may be 1, 2, or more of sulfasalazine, salicylates, mesalamine (Asacol(copyright), PandG) or Zelmac(copyright), (Bristol-Myers Squibb), which may be employed in amounts specified in the PDR or otherwise known in the art.
The agent for treating osteoporosis which may be optionally employed in combination with the DP4 inhibitor of the invention may be 1, 2, or more of alendronate sodium (Fosamax(copyright), Merck, tiludronate (Skelid(copyright), Sanofi), etidronate disodium (Didronel(copyright), PandG), raloxifene HCl (Evista(copyright), Lilly), which may be employed in amounts specified in the PDR.
In carrying our the method of the invention, a pharmaceutical composition will be employed containing the compounds of structure I, with or without another antidiabetic agent and/or other type therapeutic agent, in association with a pharmaceutical vehicle or diluent. The pharmaceutical composition can be formulated employing conventional solid or liquid vehicles or diluents and pharmaceutical additives of a type appropriate to the mode of desired administration. The compounds can be administered to mammalian species including humans, monkeys, dogs, etc. by an oral route, for example, in the form of tablets, capsules, granules or powders, or they can be administered by a parenteral route in the form of injectable preparations. The dose for adults is preferably between 10 and 1,000 mg per day, which can be administered in a single dose or in the form of individual doses from 1-4 times per day.
A typical capsule for oral administration contains compounds of structure I (250 mg), lactose (75 mg) and magnesium stearate (15 mg). The mixture is passed through a 60 mesh sieve and packed into a No. 1 gelatin capsule.
A typical injectable preparation is produced by aseptically placing 250 mg of compounds of structure I into a vial, aseptically freeze-drying and sealing. For use, the contents of the vial are mixed with 2 mL of physiological saline, to produce an injectable preparation.
DP4 inhibitor activity of the compounds of the invention may be determined by use of an in vitro assay system which measures the potentiation of inhibition of DP4. Inhibition constants (Ki values) for the DP4 inhibitors of the invention may be determined by the method described below.
Porcine enzyme was purified as previously described (1), with several modifications. Kidneys from 15-20 animals were obtained, and the cortex was dissected away and frozen at xe2x88x9280xc2x0 C. Frozen tissue (2000 -2500 g) was homogenized in 12 L of 0.25 M sucrose in a Waring blender. The homogenate then was left at 37xc2x0 C. for 18 hours to facilitate cleavage of DP-4 from cell membranes. After the cleavage step, the homogenate was clarified by centrifugation at 7000xc3x97g for 20 min at 4xc2x0 C., and the supernatant was collected. Solid ammonium sulfate was added to 60% saturation, and the precipitate was collected by centrifugation at 10,000xc3x97g and was discarded. Additional ammonium sulfate was added to the supernatant to 80% saturation, and the 80% pellet was collected and dissolved in 20 mM Na2HPO4, pH 7.4.
After dialysis against 20 mM Na2HPO4, pH 7.4, the preparation was clarified by centrifugation at 10,000xc3x97g. The clarified preparation then was applied to 300 mL of ConA Sepharose that had been equilibrated in the same buffer. After washing with buffer to a constant A280, the column was eluted with 5% (w/v) methyl xcex1-D-mannopyranoside. Active fractions were pooled, concentrated, and dialyzed against 5 mM sodium acetate, pH 5.0. Dialyzed material then was flowed through a 100 mL Pharmacia Resource S column equilibrated in the same buffer. The flow through material was collected and contained most of the enzyme activity. Active material again was concentrated and dialyzed into 20 mM Na2HPO4, pH 7.4. Lastly, the concentrated enzyme was chromatographed on a Pharmacia S-200 gel filtration column to removed low molecular weight contaminants. Purity of column fractions was analyzed by reducing SDS-PAGE, and the purest fractions were pooled and concentrated. Purified enzyme was stored in 20% glycerol at xe2x88x9280xc2x0 C.
Enzyme was assayed under steady-state conditions as previously described (2) with gly-pro-p-nitroanilide as substrate, with the following modifications. Reactions contained, in a final volume of 100 xcexcl, 100 mM Aces, 52 mM TRIS, 52 mM ethanolamine, 500 xcexcM gly-pro-p-nitroanilide, 0.2 % DMSO, and 4.5 nM enzyme at 25xc2x0 C., pH 7.4. For single assays at 10 xcexcM test compound, buffer, compound, and enzyme were added to wells of a 96 well microtiter plate, and were incubated at room temperature for 5 min. Reactions were started by addition of substrate, The continuous production of p-nitroaniline was measured at 405 nM for 15 min using a Molecular Devices Tmax plate reader, with a read every 9 seconds. The linear rate of p-nitroaniline production was obtained over the linear portion of each progress curve. A standard curve for p-nitroaniline absorbance was obtained at the beginning of each experiment, and enzyme catalyzed p-nitroaniline production was quantitated from the standard curve. Compounds giving greater than 50% inhibition were selected for further analysis.
For analysis of positive compounds, steady-state kinetic inhibition constants were determined as a function of both substrate and inhibitor concentration. Substrate saturation curves were obtained at gly-pro-p-nitroanilide concentrations from 60 xcexcM to 3600 xcexcM. Additional saturation curves also were obtained in the presence of inhibitor. Complete inhibition experiments contained 11 substrate and 7 inhibitor concentrations, with triplicate determinations across plates. For tight binding inhibitors with Kis less than 20 nM, the enzyme concentration was reduced to 0.5 nM and reaction times were increased to 120 min. Pooled datasets from the three plates were fitted to the appropriate equation for either competitive, noncompetitive or uncompetitive inhibition.
(1) Rahfeld, J. Schutkowski, M., Faust, J., Neubert., Barth, A., and Heins, J. (1991) Biol. Chem. Hoppe-Seyler, 372, 313-318.
(2) Nagatsu, T., Hino, M., Fuyamada, H., Hayakawa, T., Sakakibara, S., Nakagawa, Y., and Takemoto, T. (1976) Anal. Biochem., 74, 466-476.
The following abbreviations are employed in the Examples and elsewhere herein:
Ph=phenyl
Bn=benzyl
i-Bu=iso-butyl
Me=methyl
Et=ethyl
Pr=propyl
Bu=butyl
TMS=trimethylsilyl
FMOC=fluorenylmethoxycarbonyl
Boc or BOC=tert-butoxycarbonyl
Cbz=carbobenzyloxy or carbobenzoxy or benzyloxycarbonyl
HOAc or AcOH=acetic acid
DMF=N,N-dimethylformamide
EtOAc=ethyl acetate
THF=tetrahydrofuran
TFA=trifluoroacetic acid
Et2NH=diethylamine
NMM=N-methyl morpholine
n-BuLi=n-butyllithium
Pd/C=palladium on carbon
PtO2=platinum oxide
TEA=triethylamine
EDAC=3-ethyl-3xe2x80x2-(dimethylamino)propyl-carbodiimide hydrochloride (or 1-[(3-(dimethyl)amino)propyl])-3-ethylcarbodiimide hydrochloride)
HOBT or HOBT.H2O=1-hydroxybenzotriazole hydrate
HOAT=1-hydroxy-7-azabenzotriazole
PyBOP reagent=benzotriazol-1-yloxy-tripyrrolidino phosphonium hexafluorophosphate
min=minute(s)
h or hr=hour(s)
L=liter
mL=milliliter
xcexcL=microliter
g=gram(s)
mg=milligram(s)
mol=mole(s)
mmol=millimole(s)
meq=milliequivalent
rt=room temperature
sat or sat""d=saturated
aq.=aqueous
TLC=thin layer chromatography
HPLC=high performance liquid chromatography
LC/MS=high performance liquid chromatography/mass spectrometry
MS or Mass Spec=mass spectrometry
NMR=nuclear magnetic resonance
mp=melting point